Abstract Background Central nervous system leukemia (CNSL) is one of the major causes of the poor prognosis of childhood leukemia. We aimed to compare the sensitivity of cytomorphology (CM) and flow cytometry (FCM) in diagnosing CNSL, emphasizing the importance of FCM in the diagnosis process. Methods One-hundred-sixty-five children with newly diagnosed B-cell Acute Lymphoblastic Leukemia (B-cell ALL) were included in this study. Cerebrospinal fluid (CSF) samples were taken for routine CSF analysis, CM analysis, and FCM examination. Computed tomography scans and/or magnetic resonance imaging were performed at diagnosis. Patients with CNS2, CNS3, and traumatic lumbar puncture (TLP) at diagnosis received two additional courses of triple intrathecal injections during induction treatment. We compared the sensitivity of FCM and CM in the diagnosis of children with CNSL. Results One hundred and twenty-eight (77.58%) CSF samples were negative by either CM or FCM (CM−/FCM−), four (2.42%) were positive by both CM and FCM (CM+/FCM+), and thirty-three (20%) displayed a single positive finding by FCM (CM−/FCM+) (p = 0.044). By adding two intrathecal injections in the induction treatment, ten children with TLP+ had no CNS relapse, like those with TLP−. However, compared to CNS1 and TLP, the event-free survival (EFS) did not significantly improve in patients with CNS2 and CNS3. Moreover, CNSL status was associated with worse 3-year EFS (p < 0.05). Conclusions We have validated that FCM is more accurate in stratifying the status of the CNS compared to CM analysis. However, to improve the EFS rate of childhood leukemia, it is necessary to combine CM examination, FCM, and cranial imaging for the early diagnosis of CNSL.

目的 观察免疫性血小板减少症(ITP)患儿外周血淋巴细胞亚群比例的变化和血小板相关抗体的表达情况,探讨其诊断效能.方法 选取2020年6月至2021年6月深圳市儿童医院临床诊断为ITP患儿98例和健康儿童30例,采用流式细胞术检测淋巴细胞亚群和血小板相关抗体(PAIgG、PAIgM、PAIgA),同时比对24例患儿治疗前后血小板相关抗体的变化情况,并通过相关性分析和ROC曲线分析其与血小板数量的相关性和对ITP的诊断效能.结果 ITP患儿血小板相关抗体PAIgG、PAIgM、PAIgA阳性百分率均高于健康儿童,差异有统计学意义(P<0.01),ITP患儿治疗后血小板相关抗体相比治疗前有所下降,差异有统计学意义(P<0.05),淋巴细胞亚群中CD16+CD56+NK细胞比例低于健康儿童,差异有统计学意义(P<0.05),CD19+B细胞、CD3+T细胞、CD3+CD4+T细胞、CD3+CD8+T细胞及CD3+CD16+CD56+NKT细胞比例与健康儿童相比差异无统计学意义(P>0.05).PAIgG、PAIgM、PAIgA与血小板的数值呈现负相关,淋巴细胞各亚群的比例变化与血小板的降低无相关性.PAIgM对ITP的诊断价值最高,ROC曲线下面积(AUCROC)为0.910,cut-off值为21.5％,敏感性85％,特异性90％;血小板相关抗体联合CD16+CD56+NK细胞亚群并不能提高其诊断效能.结论 ITP患儿存在一定程度的免疫紊乱,相对于淋巴细胞亚群而言,血小板相关抗体对其诊断及预后评估更具有临床价值.

目的 研究不同强度的化疗对急性淋巴细胞白血病(ALL)儿童性腺功能激素指标的影响.方法 依据华南地区儿童ALL治疗协助组2016化疗方案(SCCLG-ALL-2016),对112例青春期前初发ALL儿童进行队列研究,采用化学发光法检测化疗前、化疗后3、6、12、18月的血清抗缪勒管激素(AMH)和抑制素B(INHB)激素水平,按照性别及危险度分型分组进行比较.结果 共纳入112例ALL患儿,男58例、女54例,中位年龄4.1(2.5～6.1)岁;高危型24例、中危型58例、低危型30例.正常对照组57例,男28例、女29例,中位年龄4.0(2.5～6.3)岁.采用重复测量方差分析发现,男童低危型、中危型、高危型之间血清INHB水平差异有统计学意义(F=3.60,P=0.036),化疗前后不同时间之间血清INHB水平差异有统计学意义(F=81.67,P<0.001),化疗时间与危险度分型对于血清INHB水平存在交互效应(F=5.12,P<0.001).女童低危型、中危型、高危型之间血清AMH水平差异无统计学意义(F=0.62,P=0.551),化疗前后不同时间之间血清AMH水平差异有统计学意义(F=21.32,P<0.001),化疗时间与危险度分型对于血清AMH水平存在交互效应(F=2.33,P=0.029).结论 尽管ALL儿童尚处于青春期前,SCCLG-ALL-2016化疗仍可导致其性腺功能激素指标改变,且高危型化疗影响程度高于低中危型,提示ALL儿童在化疗期间性腺功能存在不同程度的损伤.

目的 探讨细胞因子IL-23与IL-12对NK细胞功能的影响及可能的机制.方法 密度梯度离心法分离人外周血单个核细胞(PBMCs)或磁珠纯化NK细胞,不刺激或用IL-23或IL-12刺激,用流式细胞术和ELISA法检测NK细胞产生IFN-γ的情况;以K562或Jurkat细胞作为靶细胞,用流式细胞术检测NK细胞的杀伤功能并分析NK细胞在不同的刺激条件下杀伤相关分子的表达情况及pSTAT的表达情况.结果 与未刺激组相比,IL-23和IL-12均可以诱导NK细胞呈剂量和时间依赖方式产生IFN-γ;但IL-12而非IL-23可以增强NK细胞对靶细胞K562或Jurkat细胞的杀伤功能.进一步研究表明,IL-12而非IL-23可以诱导杀伤相关分子TRAIL及CD107a/b的表达.此外,IL-12诱导NK细胞表达更高水平的pSTAT4,而IL-23诱导NK细胞表达更高水平的pSTAT3.结论 与IL-12相比,IL-23亦可以诱导NK细胞产生细胞因子但不能增强NK细胞的杀伤功能,IL-23不能诱导杀伤相关分子TRAIL及CD107a/b的表达,IL-23可以诱导低水平的pSTAT4但高水平的pSTAT3的表达.

*These authors contributed equally to this work Abstract: Here, we report a rare case of a 12-year-old boy who was initially diagnosed with B cell lymphoblastic lymphoma (BLBL) and developed myeloid sarcoma (MS) eight months after chemotherapy. Next-generation sequencing (NGS) showed mutations of KRAS and NRAS genes in both the bone marrow and lymph node. He presented an abnormal karyotype of 46, XY, −9, der (16) t (9; 16) (q13; q12), +mar. He received chemotherapy according to the South China Children’s Leukemia Group 2016 protocol. Complete remission was achieved by the 15th day post-treatment. Eight months later and immediately prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and enlargement of bilateral testes. Pathological analysis of skin and testicular biopsies suggested the diagnosis of myeloid sarcoma (MS). Again, NGS examination showed mutations of KRAS and NRAS genes. The patient underwent haploidentical hematopoietic stem cell transplantation but unfortunately did not survive. The interval of eight-month interval between the initial disease onset and MS brings into question whether MS developed as part of the initial onset of disease or as a secondary tumor in association with chemotherapy. Thus, understanding the pathogenesis of MS involving abnormalities of lymphoid progenitors may assist in the prediction of prognosis and development of novel target therapies.

Objective: To identify gene variants and investigate clinical features of nonmuscle myosin heavy chain 9-related disease (MYH9-RD). Methods: In this retrospective study, the data of patients with MYH9-RD admitted to Shenzhen Children's Hospital from July 2017 to September 2020 were extracted. The gene variants, clinical features and laboratory tests results were summarized. Results: Among the 6 children, 4 were males and 2 were females, aged 4.0 (0.5-7.6) years. Main clinical manifestations included thrombocytopenia (6 cases), epistaxis (3 cases), petechias (2 cases), traumatic hematoma (1 case), and abnormal liver enzymes (1 case). One patient had no family history, and the other 5 cases were pedigrees. Two pedigrees (2 cases) had long-term microscopic hematuria, one pedigree (2 cases) had history of early cataract, and three pedigrees (5 cases) had chronic mild elevation of liver enzymes. Four MYH9 gene variants were found in 12 patients, including c.2104C>T(p.R702C) in exon 17, c.4270G>A(p.D1424N) in exon 31, c.5521G>A (p.E1841K) in exon 39, and c.5797C>T (p.R1933X) in exon 41. According to the family pedigrees analysis, except for the case of variant in exon 17 which was spontaneous mutation with no family history, the other variants were from their father or mother. The complete blood count results showed a decreased platelet number in these patients, and the counting results of the automated hematology analyzer were significantly lower than that of manual counting method ((33.4±17.2) × 10⁹ vs. (60.4±21.0) × 109/L,t=-5.83, P<0.05). The examination of the peripheral blood smear revealed the presence of thrombocytopenia with giant platelets and granulocyte inclusion bodies. The MYH9 gene variant (R702C) located at the N-terminus head domain of non-muscle myosin heavy chain ⅡA (NMMHC-ⅡA), which has ATPase activity, led to severe reduction of platelet number (<20×109/L) and obscure granulocyte inclusion bodies. However, higher platelet numbers (40×109-80×109/L) and obvious granulocyte inclusion bodies were observed in patients with tail-position mutations at C-terminus. Conclusions: The clinical phenotypes of MYH9-RD were variable. The mutations in certain regions of MYH9 gene were related to platelet count and granulocyte inclusion bodies. MYH9-RD should be considered in individuals with unknown etiology and persistent thrombocytopenia which is non-responsive to conventional treatment, regardless of family history. Complete blood count and blood smear morphology examinations are the first steps to screen and diagnose the disease. The laboratory should pay attention to the morphological review rules and standardized reports.

目的 鉴定1例非肌性肌球蛋白重链9(MYH9)基因相关疾病家系的致病突变.方法 调查收集先证者和家系成员病史资料,观察其临床特征和实验室检查指标.采用芯片捕获高通量测序方法检测先证者MYH9基因,确定突变位点后,对先证者和家系成员进行Sanger验证.结果 该家系三代4名患者均有鼻衄、瘀斑紫癜、外伤血肿或月经量增多病史,长期存在镜下血尿和蛋白尿.血涂片镜检均有血小板减少、巨大血小板和粒细胞异常包涵体"三联征".所有患者MYH9基因第40内含子供体剪接位点存在错义突变c.5765+2T>A(p.R1922Rfs43),且该基因突变与疾病表型共分离.结论 该家系存在MYH9基因c.5765+2T>A(p.R1922Rfs43)剪接突变,是MYH9基因相关疾病的致病突变,为国内首次报道.

Here, we report a rare case of a 12-year-old boy who was initially diagnosed with B cell lymphoblastic lymphoma (BLBL) and developed myeloid sarcoma (MS) eight months after chemotherapy. Next-generation sequencing (NGS) showed mutations of KRAS and NRAS genes in both the bone marrow and lymph node. He presented an abnormal karyotype of 46, XY, -9, der (16) t (9; 16) (q13; q12), +mar. He received chemotherapy according to the South China Children's Leukemia Group 2016 protocol. Complete remission was achieved by the 15th day post-treatment. Eight months later and immediately prior to the start of maintenance therapy, the patient developed fever, skin nodules in both upper arms, and enlargement of bilateral testes. Pathological analysis of skin and testicular biopsies suggested the diagnosis of myeloid sarcoma (MS). Again, NGS examination showed mutations of KRAS and NRAS genes. The patient underwent haploidentical hematopoietic stem cell transplantation but unfortunately did not survive. The interval of eight-month interval between the initial disease onset and MS brings into question whether MS developed as part of the initial onset of disease or as a secondary tumor in association with chemotherapy. Thus, understanding the pathogenesis of MS involving abnormalities of lymphoid progenitors may assist in the prediction of prognosis and development of novel target therapies.

目的 应用E6流式细胞仪建立检测CD34+细胞百分比和绝对计数的方法并进行验证.方法 收集2019年1月至2019年12月在深圳市儿童医院血液肿瘤科因重型β-地中海贫血行异基因造血干细胞移植的患儿89例,以移植患儿供的骨髓或外周血为研究对象,应用E6流式细胞仪建立ISHAGE法检测CD34+细胞百分比,应用"流式直接体积法"计算CD34+细胞的绝对计数,并结合白细胞手工计数对CD34+细胞绝对计数的结果进行验证.结果 结合白细胞手工计数对CD34+细胞的绝对计数结果的正确性进行验证,Pearson相关分析结果显示:供者外周血干细胞或骨髓干细胞CD34+细胞绝对计数流式结果与CD34+细胞绝对计数计算结果高度相关(r=0.93,R2=0.87,P<0.0001);两次供者外周血干细胞或骨髓干细胞白细胞计数和单个核百分比结果高度一致;同样,两次供者外周血干细胞或骨髓干细胞CD34+细胞流式计数百分比和绝对计数结果亦高度一致.结论应用E6流式细胞仪的"流式直接体积法"计算CD34+细胞绝对计数结果准确,可以作为造血干细胞移植术前计算供者外周血干细胞或骨髓干细胞CD34+细胞数目的可靠依据.

目的 探讨急性髓系白血病自然缓解的诱因和可能的发生机制.方法 回顾分析1例急性单核细胞白血病发生自然缓解患儿的临床资料,并结合相关文献探讨自然缓解的可能机制.结果 1岁4月龄男性患儿,确诊为急性髓系单核细胞白血病,未经化疗后发生骨髓自然缓解,约1年后出现髓系肉瘤复发,予行HLA半相合造血干细胞移植术,目前预后良好.回顾既往文献发生自然缓解患者大部分缓解之前有发热史,通常由感染导致,也可由药物撤除或输血导致.结论 髓系白血病发生自然缓解很少见,未来或许可以通过免疫疗法达到治疗白血病的目的.

OBJECTIVE:to explore the value of capillary electrophoresis in screening β- thalassemia of children, and to establish the cutoff values of HbA2 and HbF in our laboratory.METHODS:The data of hemoglobin capillary electrophoresis and genetic diagnosis of β- thalassemia from 886 examined children were retrospectively analyzed. The cutoff values of HbA2 and HbF were determined by ROC curve.RESULTS:The cutoff value of HbA2 screening minor β- thalassemia was 3.65%, the specificity was 0.996, and the sensitivity was 0.995. The cut-off value of HbF for screening minor β- thalassemia was 1.45%, specificity was 0.751 and sensitivity was 0.675. Thus, 1 case with codon5 (CCT→C) mutation, 1 case with SEA -HPFH β deletion, 1 case with - 28 (A→G) merger IVS-Ι-128 (T→G) double heterozygous mutations yet were found out, 1 case with 47 bp β gene missing has not yet been reported in literature.CONCLUSION:Capillary electrophoresis has more high sensitivity and specificity in the screening of β- thalassemia in children, especially for the detection of rare β- thalassemia.

目的 动态监测重型β-地中海贫血患儿异基因造血干细胞移植(allo-HSCT)术前及术后第1、2、3、6和12个月淋巴细胞亚群比例的变化,探讨淋巴细胞的恢复情况及重建特点.方法 选取行allo-HSCT的27例重型β-地中海贫血患儿,其中人类白细胞抗原(HLA)全相合移植术20例,HLA不全相合移植术7例.采用流式细胞术(FCM)动态监测患者移植前及术后1年内CD3+T细胞、CD3+CD4+T细胞、CD3+CD8+T细胞、CD4+/CD8+比值、CD19+B细胞及CD16+CD56+自然杀伤(NK)细胞比例的变化.结果 与allo-HSCT术前比较,重型β-地中海贫血患儿术后CD16+CD56+NK细胞比例恢复最快,术后第1、2、3、6个月均高于术前(P<0.05),第12个月恢复至术前水平(P>0.05).CD3+T细胞比例术后第1个月与术前比较差异无统计学意义(P>0.05),第2个月起降低且低于术前(P<0.01),第12个月仍低于术前(P<0.01).CD3+CD8+T细胞比例术后第1个月明显高于术前(P<0.01),随后逐渐下降,至术后第12个月时仍高于术前(P<0.05).CD3+CD4+T细胞比例术后明显低于术前(P<0.01),且呈缓慢回升趋势,至术后第12个月仍明显低于术前(P<0.01).CD4+/CD8+比值长期倒置,虽然呈缓慢上升趋势,但仍明显低于术前(P<0.01).CD19+B细胞比例术后第1个月明显低于术前(P<0.01),术后第2个月恢复至术前水平(P>0.05),术后第6个月和第12个月明显高于术前(P<0.01、P<0.05).术前及术后同一时间点男性、女性患儿之间及HLA全相合移植与HLA不全相合移植之间各淋巴细胞亚群比例差异均无统计学意义(P>0.05).结论 重型β-地中海贫血患儿allo-HSCT术后淋巴细胞亚群的免疫重建恢复顺序依次为CD16+CD56+NK细胞、CD3+CD8+T细胞、CD19+B细胞、CD3+T细胞、CD3+CD4+T细胞.

目的 探讨EB病毒感染传染性单核细胞增多症(EBV-IM)和嗜血细胞综合征(EBV-HPS)患儿外周血淋巴细胞亚群、免疫球蛋白及血游离EBV-DNA拷贝数的改变特点.方法 选取2015年1月至2017年12月经深圳市儿童医院血液科诊治的51例EBV感染患儿为实验组,其中EBV-IM患儿28例,EBV-HPS患儿23例,选取35例体检健康的儿童为对照组.全部受检者采用流式细胞技术检测外周血淋巴细胞、CD3+T淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞、CD19+B淋巴细胞、NK细胞的绝对计数及百分比,应用血清免疫球蛋白分析仪检测IgG、IgM、IgA水平,实时荧光定量PCR检测EBV-DNA拷贝数.结果 实验组与对照组受检者的淋巴细胞计数、淋巴细胞各亚群计数及百分比比较,差异均有显著统计学意义(P<0.01);EBV-IM组与对照组、EBV-HPS组比较,淋巴细胞计数、CD3+T淋巴细胞计数、CD8+T淋巴细胞计数及百分比升高、CD4+/CD8+值、CD19+B淋巴细胞百分比降低,差异有统计学意义(P<0.05);EBV-HPS组与对照组比较,淋巴细胞计数、CD3+T细胞计数、CD4+T淋巴细胞计数、CD19+B淋巴细胞计数及NK细胞计数降低,CD3+T淋巴细胞百分比、CD8+T淋巴细胞百分比升高,差异有统计学意义(P<0.05);EBV-IM组患儿的IgG、IgM、IgA较对照组及EBV-HPS组升高,差异有统计学意义(P<0.05),而EBV-HPS组与对照组免疫球蛋白比较,差异无统计学意义(P>0.05);EBV-HPS组较EBV-IM组EBV-DNA拷贝数升高,差异有显著统计学意义(P<0.01).结论 EBV-IM与EBV-HPS患儿外周血淋巴细胞亚群存在显著差异,各亚群细胞百分比结合绝对计数有助于两者的诊断及鉴别诊断;免疫球蛋白检测及EB病毒拷贝数分析可进一步辅助EBV-IM及EBV-HPS的鉴别诊断.

目的 探讨红细胞(RBC)计数、血红蛋白含量(Hb)、平均红细胞体积(MCV)、平均红细胞血红蛋白量(MCH)及血红蛋白A2(HbA2)在地中海贫血和缺铁性贫血(IDA)鉴别诊断中的应用价值并确立最佳截断值(cut-off值).方法 选取2015年1月至2017年1月深圳市儿童医院经地中海贫血基因和血清铁、铁蛋白确诊的地中海贫血和缺铁性贫血患儿为研究对象,其中α地中海贫血198例,β地中海贫血204例,缺铁性贫血153例,正常对照组108例.分别比较α、β地中海贫血与缺铁性贫血患儿的RBC计数、Hb、MCV、MCH及HbA2的值,采用ROC曲线评价鉴别地中海贫血与缺铁性贫血的血液学指标,找出最佳cut-off值,以cut-off值计算上述指标鉴别这两种贫血的灵敏度、特异性、预测值与准确率.结果 RBC计数、Hb、MCV、MCH及HbA2在鉴别儿童β地中海贫血与缺铁性贫血中差异有统计学意义(P<0.05),其最佳cut-off值分别为5.20×1012/L、92.50 g/L、60.25 fl、17.25 pg、2.85％;MCV、HbA2在鉴别α地中海贫血与缺铁性贫血中差异无统计学意义,RBC计数、Hb、MCH在鉴别α地中海贫血与缺铁性贫血中差异有统计学意义(P<0.05),其最佳cut-off值分别为5.25×1012/L、101.50 g/L、18.15 pg.结论 MCV、HbA2在鉴别儿童α地中海贫血和缺铁性贫血中无鉴别价值;RBC计数及Hb分别取最佳cut-off值时,单独或联合使用对鉴别α地中海贫血和缺铁性贫血具有筛查价值;HbA2在鉴别儿童β地贫与IDA中最具有意义,其灵敏度、特异度等均大于90％,可作为独立的鉴别指标,取cut-off值为2.85％.